A history of study and new records of terrestrial enchytraeids (Annelida, Clitellata, Enchytraeidae) from the Russian Far East

Abstract A list of terrestrial enchytraeids of the Russian Far East is compiled based on literature and extensive field data collected by the authors in 2019. A database has been created consisting of geographic coordinates, habitat type, species, and data source. For some species collected by the authors, barcoding using COI, 16s, and 12s rRNA genes has been performed. In total, there are at least 62 species of enchytraeids belonging to 12 genera. Seven species (Achaeta macroampullacea, Cognettia sphagnetorum, Enchytraeus dichaetus, Fridericia cusanica, Globulidrilus riparius, Marionina southerni, Mesenchytraeus gigachaetus) are reported in the Russian Far East for the first time. Cognettia sphagnetorum and F. cusanica are most probably introduced. Taxonomic and biogeographical remarks on some of the species found and differences from the original descriptions are provided. Some of the specimens may be undescribed species, but this requires a more in-depth examination. The Russian Far East, especially its southeastern part, is of great interest as a possible location for new species of enchytraeids.


Introduction
The Russian Far East (hereafter referred to as RFE) ranges in climate, from arctic deserts in the north to dry steppes and broadleaf forests in the south. In this paper, RFE is equated with the Far Eastern Federal District of Russia, which includes the Republic of Buryatia, Sakha Republic (Yakutia), Kamchatka, Khabarovsky, Primorsky, and Zabaykalsky Krais, Amur, Magadan and Sakhalin Oblasts, Chukotka Autonomous Okrug, and Jewish Autonomous Oblast. This region occupies 40.6% of the territory of Russia (Rosstat 2019). On the Pacific coast, the climate is monsoon, which is most pronounced in the south and weakens to the northeast. Summer here is moderately warm and rainy, and winter, although not very snowy, is cold, due to the spread of cold air from the interior of Siberia. More inland, the climate becomes more continental, with warm summers and very cold winters. The northern regions of the RFE, adjacent to the Arctic Ocean, are characterized by cold summers and cold winters. Landscapes also vary from mountain ranges in the northeast to the plains along the Amur River in the south. Part of the territory is represented by islands and archipelagos, such as Sakhalin, the Kuril Islands, the New Siberian Islands, and Wrangel Island (Krestov 2003). The large area of the RFE contributes to the diversity of biomes. Arctic desert and tundra are replaced to the south by a wide strip of coniferous forests and then by forest-steppes and steppes. In the southeast of the RFE, in Primorsky Krai, broadleaf and mixed forests are represented. Diversity of biomes leads to great diversity of flora and fauna, especially that of invertebrates (Storozhenko et al. 2002;Ryabinin 2015). However, general knowledge about the soil biodiversity of this area is rather poor and fragmentary.
The Enchytraeidae are a family of terrestrial oligochaetes consisting mainly of small unpigmented annelid worms. Enchytraeids inhabit all continents and are perhaps the most widespread representatives of the Clitellata (Erséus 2005). An ecologically plastic group, enchytraeids are found in a wide range of habitats. Various species of the family are broadly represented in soil and in the splash zone on seacoasts; they are also found in freshwater and marine sediments (Erséus and Rota 2003;Erséus 2005). Twenty of the 35 valid genera of enchytraeids are found in soil (Coleman et al. 2018;Timm and Erséus 2020), where they are important in the decomposition of organic matter (Römbke et al. 2005).
At the same time, the RFE, as well as the territory of Russia as a whole, the enchytraeid fauna has been poorly studied, and this situation has persisted since the 1980s (Nurminen 1982). Thus, researching enchytraeid diversity of the RFE has both inventory and biogeographic significance.
In the last decade, many studies have been conducted on the fauna of enchytraeids of the territories geographically close to the RFE: Manchuria (Chen andXie 2015), South Korea (Dózsa-Farkas andHong 2010;Dózsa-Farkas et al. 2015), and Japan (Torii 2012). In contrast, there is a knowledge gap for enchytraeids in the RFE itself, some studies have been conducted. Some species were described from materials collected by Russian polar expeditions at the beginning of the 20 th century (Michaelsen 1901(Michaelsen , 1916Cejka 1910Cejka , 1914. After many years, Piper et al. (1982) reported the enchytraeid fauna at several points on the coast of northeastern Russia. Nurminen (1980) found several new species of enchytraeids in samples sent from Primorsky Krai. Ganin (1984) suggested the possibility of finding even more new species in the RFE. In 1994, a Swedish-Russian expedition took samples, including for enchytraeid extraction, in the Palaearctic tundra. Some of their sampling sites were located in the RFE (Christensen and Dózsa-Farkas 1999).
In the summer of 2019, we have collected much material from across the RFE, between the city of Magadan (northern coast of the Okhotsk Sea) and the southernmost areas of Primorsky Krai. The identity of some species was confirmed with the help of molecular analysis of the genes COI, 12S rRNA, and 16S rRNA.
In this study we summarize currently available data on the fauna of terrestrial enchytraeids of the RFE, both from the literature and our own new data.

Materials and methods
To estimate terrestrial enchytraeid diversity of the RFE, a database has been created. It includes the following attributes: geographic coordinates of sampling sites, habitat type, species, and data source. Data on enchytraeids from marine and freshwater environments were not included.
Data from literature. We included data collected by other researchers. These data are mostly from tundra and taiga habitats (see below). Although there are numerous reports of enchytraeids in soil samples in the soil-zoological literature, we only included those studies where enchytraeid species were identified.
Our data. We collected our material during July and August 2019 in Khabarovsky, Primorsky, and Zabaykalsky krais, as well as Amur, Magadan, and Sakhalin oblasts (Fig. 1). The investigations were carried out in steppes, broadleaf and coniferous forests, and azonal habitats like floodplains and meadows. Within each locality (see Fig.  1), five samples each 5 cm in diameter were collected and brought to the laboratory in Moscow. They were stored at 4 °C until the extraction. Enchytraeids were extracted from soil using the wet funnel method (Römbke 1995), identified in vivo, and then preserved in 96% alcohol.
Species were identified according to Schmelz and Collado (2010) or, for species not found in Europe, by comparison with the original descriptions. Taxonomy follows Schmelz andCollado (2012, 2015), excluding genera and species described after 2015.
Some samples were selected for genetic analysis. The DNA extraction was performed using "ExtractDNA blood" kits (Evrogen, Russia). In each sample the entire enchytraeid body was used for DNA extraction. We amplified one or two of three mitochondrial regions: 12S rRNA, 16S rRNA, and cytochrome oxidase I (COI) genes. The COI fragment is the standard DNA barcode for animals (Hebert et al. 2003). The PCR mixture contained 1-3 ng of the DNA matrix, 0.1 μM of each primer, and the precast PCR mixture from Evrogen (Russia) according to the manufacturer's instructions. The primers were synthesized by Evrogen (Russia) as well. For the COI gene the primers were LCO1490 (Folmer et al. 1994)  and COI-E (Bely and Wray 2004) (5'-TATACTTCTGGGTGTCCGAAGAATCA-3') and the amplification program was: 95 °C for 5 min, 35 cycles of 95 °C for 40 sec, 45 °C for 45 sec, 72 °C for 60 sec, and finally, 72 °C for 8 min, resulting in a fragment of about 608 bp. For the 12S rRNA gene the primers were 12S-E1 (5'-AAAACATG-GATTAGATACCCRYCTAT-3') and 12SH (5'-ACCTACTTTGTTACGACT-TATCT-3') (Jamieson et al. 2002), and the amplification program was: 94 °C for 2 min; 35 cycles of 94 °C for 15 sec, 50 °C for 15 sec, 72 °C for 90 sec, and finally 72 °C for 5 min, resulting in a fragment of about 322 bp. For the 16S rRNA gene the primers were 16SAR-L (5'-CGCCTGTTTATCAAAAACAT-3') and 16SBRH (5'-CCG-GTCTGAACTCAGATCACGT-3') (Palumbi et al. 2002), and the amplification program was: 95 °C for 5 min, 35 cycles each of 95 °C for 30 sec, 50 °C for 30 sec, 72 °C for 60 sec, and finally, 72 °C for 8 min resulting in a fragment of about 423 bp.
The phylogenetic analysis for species of Fridericia Michaelsen, 1889 was conducted using the maximum likelihood (ML) method. Enchytraeus albidus Henle, 1837 was chosen as an outgroup. Phylogenetic analysis involved 25 16S rRNA nucleotide sequences; 20 of them were obtained from NCBI GenBank. The sequences were aligned using Clustal Omega (Sievers et al. 2011). There was a total of 409 positions in the final alignment. GTR model of nucleotide substitution with Gamma distribution and invariable sites was chosen using the BIC criterion (Nei and Kumar 2000). The initial tree was obtained automatically by applying NJ and BioNJ algorithms and then the topology with superior log likelihood value was selected. All positions containing gaps and missing data were eliminated. Bootstrap replications value was set to 10000. The ML analysis was performed in MEGA X (Kumar et al. 2018).
The phylogenetic analysis for species of Bryodrilus Ude, 1892 was conducted using the Bayesian inference (BI) method. Henlea perpusilla Friend, 1911 was chosen as an outgroup. Phylogenetic analysis involved 10 COI mDNA nucleotide sequences; nine of them were obtained from NCBI GenBank. The sequences were aligned using Clustal Omega (Sievers et al. 2011). There was a total of 528 positions in the final alignment. GTR model of nucleotide substitution with Gamma distribution was chosen in TOPALi v. 2 (Milne et al. 2009) using the AIC criterion (Akaike 1974). The analysis was run with 4 chains for 20,000,000 generations, sampling each 1000 generations and burning in 25%. The BI analysis was performed using MrBayes v. 3.2 software package (Ronquist et al. 2012). A list of all DNA sequences originally obtained in this study is provided ( Table 1).

List of localities with confirmed enchytraeid data
The habitats are described and the geographical coordinates (WGS 84) are provided with varying degree of precision depending on the information given in the original source. For the sites based on literature data, the respective references are given. The absence of a literature reference presumes that enchytraeids from this site were identified by us. In the absence of an accurate geographical reference (literature from the beginning of the 20 th century), a point on the map (Fig. 1) shows only approximate place of origin of the sample. In the commented list of terrestrial enchytraeids of the RFE, the localities are given for each species, in accordance with description of the sampling sites.

Results
We have found at least 12 genera and not fewer than 62 species in the RFE. No enchytraeids were found in sites [14] and [15], possibly due to the aridity of steppe habitats. The enchytraeid fauna consisted mostly of species from genera Mesenchytraeus Eisen, 1878 (15 species [20,23,24,26,27,28,31]. For single records of species without a clear geographical reference, the approximate location is indicated directly in the list. Remarks on some species are also given below.

A commented list of terrestrial Enchytraeidae of the Russian Far East
Genus Achaeta Vejdovský, 1878 1. Achaeta macroampullacea Dózsa-Farkas, Felföldi, Nagy & Hong, 2018 -29, 31. Specimens are fully consistent with the original description. Described from Jeju Island, Korea (Dózsa-Farkas et al. 2018), this is the first find of the species outside the original description.

Enchytraeus dichaetus
21. Fridericia callosa (Eisen, 1878) -9, 10, 11, 13.   Schmelz, 2003 -27. This species was described from Germany (Schmelz 2003) and is known from southern Europe (Schmelz and Collado 2010), but recently it was also recorded in Tibet . We found it from one of Kuril Islands. Thus, it is possibly a Holarctic species. This is the first report of F. cusanica in Russia.

Fridericia cusanica
23. Fridericia cf. cusanicaformis Dózsa-Farkas, Feldföldi & Hong, 2015 -26, 29. Differences from the original description (Dózsa-Farkas et al. 2015) are the absence of the spermathecal ectal gland and only one chaeta in ventral post-clitellar bundles. Our specimens were not fully mature, so these differences cannot be considered taxonomically significant. This is the first finding outside of Korea.
24. Fridericia cf. schmelzi Cech & Dózsa-Farkas, 2005 -26. The specimens from Primorsky Krai differed from the original description in the number of preclitellar nephridia (four). They had mostly three chaetae in lateral preclitellar bundles. The great distance to previously recorded localities (Hungary, Canada; Schmelz and Collado 2010) also raise doubts about the species' identity.
25. Fridericia sp. A -29. This is a new species. It will be described in an upcoming article. 26. Fridericia sp. B -25. This species does not fit the descriptions of the other species of Fridericia found in the RFE. This is a rare species, and more specimens needed for proper determination.
The specimens possess up to three chaetae in the ventral preclitellar bundles. This is the first find of this species and the genus in a terrestrial habitat in Russia. Globulidrilus riparius is possibly a trans-Palaearctic species having a range from Europe (Schmelz and Collado 2010) to Japan (Torii 2012). It was recently discovered than G. riparius is a complex of several species (Martinsson and Erséus 2018), but these are still undescribed. Cernosvitov, 1934 28. Hemienchytraeus sp. A -26. This is the first find of the genus in Russia. The taxonomic status was not determined due to the poor condition of the extracted specimens.
42. Marionina argentea (Michaelsen, 1889) s.l. -9. Christensen and Dózsa-Farkas (1999) wrote that the morphology of their specimens from the RFE is consistent with that accepted by Nielsen and Christensen (1959) before the separation of M. argentea into four species (Rota 2013). Thus, we use sensu lato approach here.

Discussion
Representatives of Mesenchytraeus were found only in taiga forests and tundra. Thus, the biogeographic border between coniferous and deciduous forests, which runs across the RFE (Ryabinin et al. 2018), can also be the southern border of the unique Beringian fauna of enchytraeids, characterized by the dominance of large Mesenchytraeus . The RFE is apparently located at the contact area of three large enchytraeid faunas: (i) the Beringian fauna, (ii) the Euro-Siberian fauna, characterized by a great variety of Fridericia and Henlea spp. and the presence of small Enchytraeus spp., part of the Enchytraeus buchholzi-group (Nurminen 1973;Schmelz and Collado 2010), and (iii) the East-Asian fauna, characterized by a large number of endemic species (especially Hemienchytraeus spp.) (Dózsa-Farkas and Hong 2010) and possibly a few representatives of the boreal genus Cognettia ). Thus, biogeographically, enchytraeids of the RFE should be studied not as single fauna of the whole region, but as a combination of several, partially overlapping faunas.
A list of enchytraeids of the RFE is prepared for the first time and consists of at least 62 species. This number is about half the number of species in Germany (Römbke et al. 2013), mainly because of the lack of knowledge of the RFE. Thus, this article is just a first step, with some unaddressed questions on the enchytraeid fauna for future studies. Some species, especially those that were described in the 1900-1910s, need taxonomic revision because their descriptions are very poor and inconsistent with modern science. Based on our molecular results, we suggest two potentially new species. Undeniably, the Russian Far East, especially its southeastern part, is of great interest as a possible location for new species of enchytraeids.